Vectors and process for producing high purity 6,12-dideoxyerythromycin A by fermentation

ABSTRACT

A process for producing high purity 6,12-dideoxyerythromycin A using recombinant DNA technology is disclosed. The erythromycin producing strain, Saccharopolyspora erythraea, lacking the erythromycin C-12 and C-6 hydroxylases produces a mixture of 6,12-dideoxyerythromycin A and the precursor molecule, 6-deoxyerythromycin D. To achieve conversion of the precursor to the final product, a second copy of eryG is inserted into a non-essential region of the Sac. erythraea chromosome resulting in high purity 6,12-dideoxyerythromycin A.

This application claims the benefit of U.S. Provisional application No.60/001,835, filed Aug. 3, 1995.

TECHNICAL FIELD

The present invention relates to the production of an erythromycinderivative. In particular, the present invention relates to theproduction of high purity 6,12-dideoxyerythromycin A through geneticmanipulation of the producing organism.

BACKGROUND OF THE INVENTION

Erythromycin A is a clinically useful, broad-spectrum macrolideantibiotic produced by the gram positive bacterium, Saccharopolysporaerythraea (Sac. erythraea) Intermediates of erythromycin biosynthesis,which may be useful in the design and development of new drugs, areproduced in minute quantities by Sac. erythraea and occur as mixtureswith other erythromycin derivatives, complicating chemical modificationsof these compounds.

As taught in the art, (see Donadio et al., Genetics and MolecularBiology of Industrial Microorganisms, eds. C. L. Hershberger, S. W.Queener, and G. Hegeman, 1989, American society for Microbiology,Washington, D.C. 20005) the biosynthesis of erythromycin A by Sac.erythraea, is achieved according to the proposed right-hand pathwayshown in FIG. 1. The 14-membered macrolactone, 6-deoxyerythronolide B,is first made from propionyl and 2-methylmalonyl thioesters and is thenhydroxylated at the C-6 position to form erythronolide B. The sugarsmycarose and desosamine are synthesized from glucose and are added toerythronolide B to make erythromycin D. The next steps in the proposedpathway are (in either order) the hydroxylation of erythromycin D at theC-12 position (resulting in the formation of erythromycin C) ormethylation of the C-3" position (resulting in the formation oferythromycin B). Subsequent hydroxylation of erythromycin B ormethylation of erythromycin C produces erythromycin A.

Our present understanding of the genes responsible for the biosynthesisof erythromycin and techniques to inactivate genes in Sac. erythraeaallow the directed manipulation of the pathway in order to produceprecursors and derivatives of erythromycin A. Naturally occurringprecursors of erythromycin A, such as erythromycin B and erythromycin Dare readily produced by these methods. However, other attempts to makehighly pure derivatives of erythromycin A in vivo are not alwayssuccessful, especially when alterations are made which change thesubstrates for enzymes acting in later stages of biosynthesis. It is incases where further genetic modifications may become necessary.

SUMMARY OF THE INVENTION

The method of the present invention includes the genetic modification ofan erythromycin producing microorganism, so that it is transformed intoa strain which produces high purity 3"-O-methylated erythromycinderivatives. In particular, a non-essential region of the chromosomalDNA is genetically modified by the insertion of a second copy of eryG,whose product is the erythromycin 3"-O-methyltransferase, and whichnormally converts erythromycins D and C into erythromycins B and A,respectively.

A microorganism embodying the present invention is a novel strain ofSac. erythraea which, upon cultivation in an aqueous medium, produceshigh purity 6,12-dideoxyerythromycin A of the formula: ##STR1##

Transformation of an erythromycin-producing microorganism into a6,12-dideoxyerythromycin A producing strain is accomplished by mutagenictechniques, and in particular, through gene replacement by homologousrecombination. Using this methodology, the eryF and eryK genes, whichencode the cytochrome P-450 enzymes essential for hydroxylatingerythromycin at the C-6 and C-12 positions, respectively, are replacedby integrative plasmids which carry deletions in these genes. As aresult of replacing the wild type genes with the deleted copies, neitherthe C-6 nor C-12 positions are hydroxylated. As shown theoretically inthe left hand side of FIG. 1, a deletion in the eryF gene prevents theconversion of 6-deoxyerythronolide B to erythronolide B; the addition ofthe sugar groups results then in the formation of 6-deoxyerythromycin D.The second deletion mutation, i.e. in the eryK gene, preventshydroxylation of 6-deoxyerthromycin D to 6-deoxyerythromycin C. Thus, inthe absence of a functional eryK gene, methylation of 6-deoxyerthromycinD results directly in the formation of 6,12-dideoxyerthromycin A (whichmay also be designated as 6-deoxyerythromycin B).

However, a complicating factor in the formation of a6,12-dideoxyerthyromycin A producing strain is that 6-deoxyerythromycinD serves a poor substrate for the erythromycin 3"-O-methyltransferase,which converts the substrate to 6,12-dideoxyerythromycin A. This resultsin a low ratio of the desired 6,12-dideoxyerythromycin A product to6-deoxyerythromycin D, the precursor. Thus an additional requirement forthe production of high purity 6,12-dideoxyerythromycin A is theintroduction of a second copy of the gene, eryG, which encodes the 3"-O-methyltransferase, into the producing organism. In this particularembodiment of the invention, a plasmid was constructed which allowed asecond copy of eryG, driven by the ermE* promoter, to be inserted viahomologous recombination into a non-essential region of the Sac.erythraea chromosome and to be stably maintained in the Sac. erythraeastrain.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be more readily appreciated in connectionwith the accompanying drawings, in which:

FIG. 1 is a proposed metabolic pathway for the biosynthesis oferythromycin A (on the right-hand side) and 6,12-dideoxyerythromycin Ain Sac. erythraea; (on the left-hand side);

FIG. 2a and b are flow diagrams depicting the construction of pDPE4;

FIG. 3 is a flow diagram depicting the construction of pGM504;

FIG. 4a-c are flow diagrams depicting the construction of pDPE35;

FIG. 5 is a schematic representation of gene replacement in Sac.erythraea;

FIG. 6 is the thin layer chromatography of the products of thefermentation of ER720-KF;

FIG. 7 is the thin layer chromatography of the products of thefermentation of ER720-KFG+;

FIG. 8a illustrates the amounts of 6,12-dideoxyerythromycin A and6-deoxyerythromycin D produced in a genetically engineered Sac.erythraea strain ER720-KF;

FIG. 8b illustrate the amounts of 6,12-dideoxyerythromycin A and6-deoxyerythromycin D produced by the genetically engineered Sac.erythraea strain ER720-KFG+;

FIG. 9a and b are flow diagrams depicting the construction of pKAS37.

FIG. 10 depicts a restriction map of pKAS37.

FIG. 11a and b are flow diagrams depicting the construction of pKASI37.

FIG. 12 depicts a restriction map of pKASI37.

FIG. 13 depicts the single stranded DNA sequence of a fragment of DNAcontained within the second site region.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for the genetic modification oferythromycin producing microorganisms which enables them to producehighly pure erythromycin derivatives having -O -methylation at the 3"position of the molecule. The compounds of the present invention include6,12-dideoxyerythromycin A which is represented by the structuralformula: ##STR2## This compound was obtained by growing the geneticallymodified erythromycin producing microorganism in liquid culture and thenextracting the compound from the culture medium; the compound was foundto be the dominant erythromycin derivative in the fermentation.

The present invention provides a method for the preparation of thehighly pure 3"-O-methylated erythromycin derivatives, which comprisestransforming an erythromycin producing strain into a variant producingthe desired compound. In one embodiment of the invention, theerythromycin producing microorganism is the bacterium, Sac. erythraea.Following genetic manipulation, the resulting transformant is not onlydeficient in the cytochrome P-450 enzymes encoded by eryF and eryK butalso contains an additional copy of eryG, which encodes the erythromycin3"-O-methyltransferase. It is the presence of this second copy of eryGwhich allows efficient conversion of 6-deoxyerythromycin D to6,12-dideoxyerythromycin A, and in so doing, provides a strain of Sac.erythraea which produces high purity end product rather than a mixtureof erythromycins as shown in FIGS. 8a and 8b.

The present invention also provides, as an example, a particular methodfor the introduction of the second copy of eryG into a non-essentialregion of the Sac. erythraea chromosome, comprising replacement, byhomologous recombination, of a section of that non-essential region ofthe chromosome with a copy of the same region containing embeddedtherein a thiostrepton resistance marker and a second copy of eryG whichis under the control of the ermE* promoter.

The methods of the present invention are widely applicable toerythromycin-producing microorganisms, including but not limited toSaccharopolyspora species, Streptomyces griseoplanus, Nocardia sp.,Micromonospora sp., Arthrobacter sp. and Streptomyces antibioticus. Ofthese, Sac. erythraea is the most preferred. Of course, the specificsequence of the homologous second site in a non-essential region of adifferent microorganism may vary somewhat from that shown in SEQ. ID.NO.:1 for Sac. erythraea but the method of identifying such a site iswithin routine skill of those practicing the art.

Both the C-6 and C-12 hydroxylations are catalyzed by cytochrome P-450enzymes encoded for by the eryF and eryK genes, respectively. Anerythromycin-producing Sac. erythraea strain which lacks these twoactivities would be predicted to produce 6,12-dideoxyerythromycin A. Onemeans of eliminating these hydroxylation reactions is through adisabling mutation of the cellular genes required for the operation ofthe cytochrome P-450 monooxygenase system. This can be accomplished byreplacing these genes with copies containing deletions, thereby makingthe genes non-functional and non-revertable. Any plasmid designed forgene replacement by homologous recombination which disrupts thehydroxylation steps in erythromycin biosynthesis can be utilized.Furthermore, the method of the present invention is in no way limited tothe use of gene replacement to produce mutants defective in C-6 and C-12hydroxylation of erythromycin. Other systems which disrupt thehydroxylase systems, such as gene disruption, transposon mutagenesis orchemical or light induced mutagenesis, can be used to produce thedesired genetic modification of the microorganism. Such alternativeprocedures are well known to those of ordinary skill in the art.

Although several methods are known in the art for inserting foreign DNAinto a plasmid to form a gene replacement plasmid, the method preferredin accordance with this invention is shown schematically in FIGS. 2 and3 and demonstrated in the Examples below. In a preferred embodiment ofthe present invention, selectable DNA plasmids are constructed whichcomprise (a) a fragment of plasmid pIJ702 or pIJ486 containing an originof replication and a fragment of DNA conferring resistance to theantibiotic thiostrepton (tsr), each of which are functional inStreptomyces; (b) an origin of replication and a DNA fragment conferringresistance to the antibiotic ampicillin (amp), each of which arefunctional in E. coli, and (c) a DNA fragment from the Sac. erythraeachromosome containing the mutated (i.e. deleted) gene of interest and atleast about 1 kb of contiguous DNA flanking both sides of the mutatedgene, each of which is capable of acting as a recognition sequence forplasmid integration and subsequent excision of the plasmid from thegenome. If the excision event occurs on the side of the deletionopposite of that of the integration event, the wild type gene will bereplaced by the deleted one, as schematically illustrated in FIG. 5.Example 1 and FIG. 2 are examples of a plasmid constructed for creatinga deletion in eryK. Example 2 and FIG. 3 are examples of a plasmidconstructed for creating a deletion in eryF.

The particular antibiotic resistance genes and functional origins ofreplication identified above are necessary only inasmuch as they allowfor the selection and replication of the desired recombinant plasmids.Other functional markers and origins of replication may also be used inthe practice of the invention. Likewise, any recognition sequence may beused which enables the recombinant plasmid to be integrated into aportion of the genome adjacent to the gene of interest and excise on theother side of the gene of interest in order to replace that gene with amutated copy. In addition, the plasmid of the invention may beconstructed without the use of a partial genomic digest, as in the aboveexamples. Instead, if the sequences of the regions flanking eryF anderyK are known, a recognition sequence may be synthesized de novo (forexample by polymerase chain reaction) and ligated with the necessaryorigin and resistance fragments to form the gene replacement plasmids.

In Example 4, an erythromycin producing strain of Sac. erythraea wasgenetically modified to be deficient in the erythromycin C-6 and C- 12hydroxylases. This was accomplished by first replacing the wild typecopy of eryK (encoding the C-12 hydroxylase) with a deleted copy usingplasmid pDPE4, described in Example 1. As predicted from the proposedpathway for erythromycin biosynthesis, the mutant strain producederythromycin B, with some erythromycin D also being produced early inthe fermentation. The eryF gene (encoding the C-6 hydroxylase) of thismutant strain was then replaced by a deleted copy of the gene usingplasmid pGM504, described in Example 2. The expected product of thisdoubly deleted strain, 6,12-dideoxyerythromycin A, was made but thestrain also produced large amounts of 6-deoxyerythromycin D throughout asix day fermentation, with the 6-deoxyerythromycin D being the dominantderivative from days 1 to 6, as shown in FIG. 6.

In order to produce highly pure 6,12-dideoxyerythromycin A, an extracopy of eryG was introduced into a non-essential region of the Sac.erythraea chromosome. The product of eryG is the 3"-O-methyltransferasewhich normally converts erythromycins D and C to erythromycin B and A,respectively. The preferred method for constructing a gene replacementplasmid for the addition of a second copy of eryG into the Sac.erythraea chromosome is shown schematically in FIG. 4 and described inExample 3. In a preferred embodiment of the present invention, aselectable DNA plasmid is constructed which comprises (a) a fragment ofplasmid pCD1 containing an origin of replication functional inStreptomyces and Saccharopolyspora; (b) an origin of replication and aDNA fragment conferring resistance to the antibiotic ampicillin, each ofwhich are functional in E. coli; and (c) a DNA fragment from a region ofthe Sac. erythraea chromosome of unknown, but non-essential functioncapable of acting as a recognition site for plasmid integration andexcision. (Hereafter, this DNA fragment of unknown but non-essentialfunction is referred to as the `second site` region. FIG. 13 depictsapproximately 1 kb of sin stranded DNA sequence which is a portion ofthe second site region). Embedded within the `second site` region aretwo additional DNA fragments, one encoding 3"-O-methyltransferaseoperably linked to the ermE* promoter, and a second fragment fromplasmid pWHM3 (also referred to herein as pCS5) which confers resistanceto the antibiotic thiostrepton. A culture of E. coli DH5α which containsa plasmid embodying the invention, designated pDPE35, has been depositedwith the Agricultural Research Culture Collection, Peoria, Ill. underthe terms of the Budapest Treaty and has been accorded the accessionnumber NRRL B-21486.

As in the previous Examples, the particular antibiotic resistance genesand functional origins of replication identified above are necessaryonly inasmuch as they allow for the selection and replication of thedesired recombinant plasmid. Other markers and origins of replicationmay be used. Likewise, any DNA fragment which is homologous to anon-essential region of the Sac. erythraea chromosome may be used as theintegration/excision recognition sequences surrounding the eryG and tsrgenes. Example 5 describes the use of pDPE35 in the construction of astrain of Sac. erythraea, which had previously been deleted in eryK anderyF, and which now contains an extra copy of eryG This extra copy oferyG allows the production of high purity 6,12-dideoxyerythromycin Aover at least a 4 day fermentation period, as shown in FIG. 7. The Sac.erythraea strain having deletions in eryK and eryF, referred to hereinas strain ER720-KFG+, has been deposited with the Agricultural ResearchCulture Collection, Peoria, Ill. under the terms of the Budapest Treatyand has been accorded accession number NRRL 21484.

A. DEFINITIONS

The following words and phrases have the meaning set forth below.

The term "cytochrome P-450 monooxygenase system" as used herein refersto a group of proteins (two flavoproteins, an iron-sulphur protein andthe C-6 or C-12 hydroxylase enzymes) which function together to causehydroxylation of erythromycin B or its derivatives in Sac. erythraea.The term "cytochrome P-450 enzymes" refers to the C-6 or C-12hydroxylase enzymes of the cytochrome P-450 monooxygenase system.

The term "erythromycin derivative" as used herein refers to anyerythromycin-like compound having antibiotic and/or prokinetic activity.Erythromycin-like compounds are typically characterized by having a14-membered macrolactone ring and two O-linked sugar molecules, such asare found in erythromycins A, B, C and D. "Erythromycin derivatives" areintended to include erythromycin -like compounds having modificationsand/or substituents in the macrolactone ring and/or sugar portions,provided they serve as substrate for 3"-O-methyl transferase. Forexample, common known modifications include:

4" deoxyerythromycin;

6-deoxyerythromycin D;

6,9 epoxyerythromycin;

6-O-methylerythromycin;

4"-amino-6,4"-dideoxyerythromycin A;

9,4"-diamino-6,9,4"-trideoxyerythromycin A;

8,9-anhydro-4"-deoxyerythromycin A-6,9-hemiketal;

8,9-anhydro-4"-deoxyerythromycin B-6,9-hemiketal;

8,9-anhydro-4"-deoxy-3'-N-desmethylerythromycin A-6,9-hemiketal;

8,9-anhydro-4"-deoxy-3'-N-desmethyl-3'-N-ethylerythromycinA-6,9-hemiketal;

8,9-anhydro-4"-deoxy-3'-N-propargylerythromycin A-6,9-hemiketal bromide;

8,9-anhydro-4"-deoxy-3'-N-desmethylerythromycin B-6,9-hemiketal;

8,9-anhydro-4"-deoxy-3'-N-desmethyl-3'-N-ethylerythromycinB-6,9-hemiketal;

8,9-anhydro-4"-deoxy-3'-N-propargylerythromycin B-6,9-hemiketal bromide;

9-deoxo-4 ",6-dideoxy-8-epi-6,9-epoxyerythromycin A;

9-deoxo-3'-N-desmethyl-4",6-dideoxy-8-epi-6,9-epoxyerythromycin A;

9-deoxo-3'-N-desmethyl-4",6-dideoxy-8-epi-3'-N-ethyl-6,9-epoxyerythromycinA;

9-deoxo-4",6-dideoxy-8-epi-6,9-epoxy-3'-N-propargylerythromycin Abromide;

9-deoxo-4 ",6-dideoxy-6,9-epoxyerythromycin A;

9-deoxo-3 '-N-desmethyl-4",6-dideoxy-6,9-epoxyerythromycin A;

9-deoxo-3'-N-desmethyl-4",6-dideoxy-6,9-epoxy-3'-N-ethylerythromycin A;and

9-deoxo-4",6-dideoxy-6,9-epoxy-3'-N-propargylerythromycin A bromide.

The term "expression" as used herein refers to the combination ofintracellular processes, including transcription and translationundergone by a coding DNA molecule such as a structural gene to producea polypeptide.

The term "homologous recombination" as used herein refers tocomplementary base-pairing and crossing over between DNA strandscontaining identical or nearly identical sequences.

The terms "origin of replication" as used herein refers to a DNAsequence that controls and allows for replication and maintenance of aplasmid or other vector in a host cell.

The term "operably linked" as used herein refers to the control exertedby the promoter over the initiation of transcription of a structuralgene.

The term "promoter" as used herein refers to a recognition site on a DNAsequence or group of DNA sequences that provide an expression controlelement for a structural gene and to which RNA polymerase specificallybinds and initiates RNA synthesis (transcription) of that gene.

The term "restriction fragment" as used herein refers to any linear DNAgenerated by the action of one or more restriction enzymes.

The term structural gene refers to a gene that is expressed to produce apolypeptide.

The term "transformation" as used herein refers to a process ofintroducing an exogenous DNA sequence (e.g. a vector, a recombinant DNAmolecule) into a cell or protoplast in which that exogenous DNA isincorporated into a chromosome or is capable of autonomous replication.

The term "vector" as used herein refers a DNA molecule capable ofreplication in a host cell and/or to which another DNA segment can beoperatively linked so as to bring about replication of the attachedsegment. A plasmid is an exemplary vector.

B. BACTERIAL STRAINS, PLASMID VECTORS, AND GROWTH MEDIA

The erythromycin-producing microorganism used to practice the followingexamples of the invention was Sac. erythraea ER720 (DeWitt, J. P. J.Bacteriol. 164: 969 (1985)). The host strain for the growth of E. coliderived plasmids was DH5α from Bethesda Research Laboratories (BRL),Gaithersburg, Md.

Plasmid pUC18, pUC19 and pBR322 can be obtained from BRL. Plasmid pCS5,is a multifunctional vector for integrative transformation of Sac.erythraea. (Plasmid pCS5 has been described by Vara, et al. J.Bacteriol. 171(11): 5872 (1989) and was originally designated as pWHM3).Plasmids pIJ702 (described by Katz, et al. J. Gen. MicrobioL. 129:2703.(1983)) and pIJ4070 were obtained from the John Innes Institute. PlasmidpCD1 was obtained from Claude Dery, University of Sherbrook, Quebec,Canada. Restriction map analysis and partial sequencing have shown thisplasmid to be related to pJV1 described by Doull, J. L. et al. FEMSMicrobiol. Lett. 16: 349 (1983).

Sac. erythraea was grown for protoplast transformation and routineliquid culture in 50 ml of SGGP medium (Yamamoto, et al., J. Antibiot.39:1304 (1986)), supplemented with 10 micrograms/milliliter (μg/mL) ofthiostrepton for plasmid selection where appropriate.

C. REAGENTS AND GENERAL METHODS

Commercially available reagents were used to make compounds, plasmidsand genetic variants of the present invention, including ampicillin,thiostrepton, (purchased from Sigma Chemical Co., St .Loius, Mo.)restriction endonucleases, T4-DNA ligase, and calf intestine alkalinephosphatase (CIAP) (purchased from New England Biolabs, Beverly, Mass.).

Standard molecular biology procedures (Maniatis, et al., MolecularCloning A Laboratory Manual, Cold Spring Harbor Laboratory (1982)) wereused for the construction and characterization of integrative plasmids.Plasmid DNA was routinely isolated by the alkaline lysis method(Birnboim, H. C. and Doly, J., Nucleic Acids Res. 7:1513 (1979)).Restriction fragments were recovered from 0.8-1% agarose gels witheither Prep-A-Gene (BioRad, Hercules, Calif.) or Gene Clean II (Bio101,Vista Calif.). The products of ligation for each step of plasmidconstructions were used to transform the intermediate host, E. coli DH5α(purchased from BRL), which was cultured in the presence of ampicillinto select for host cells carrying a recombinant plasmid. Screening forthe presence of insert DNA with X-gal was used where appropriate.Plasmid DNAs were isolated from individual transformants that had beengrown in liquid culture and were characterized with respect to knownrestriction sites.

Integrative transformation of Sac. erythraea protoplasts, and routinegrowth and sporulation were carried out according to proceduresdescribed in Donadio, et al., Science 115:97 (1991), Weber and Losick,Gene 68:173 (1988) and Yamamoto, et al., J. Antibiot. 39:1304 (1986).

The following abbreviations are used throughout the application:

a. TES: N-tris(Hydroxymethyl)methyl-2-aminoethane-sulfonic acid

b. R3M: A growth medium containing per 1 liter aqueous solution: 103 gsucrose, 0.25 g K₂ SO₄, 4 g yeast extract, 4 g casamino acids, 4 gtryptone, 22 grams agar in 830 mL of H₂ O. The solution is sterilized byautoclaving. After sterilization, the following additional ingredientsare added: 20 mL of 2.5 M MgCl2, 20 mL of 50% glucose, 20 mL of 2.5MCaCl₂, 12.5 mL of 2M Tris-HCI, pH 7.0, 2 mL of trace elements solution(Hopwood, et al, 1985, Genetic Manipulation of Streptomyces A LaboratoryManual, The John Innes Institute), 0.37 mL of 0.5M KH₂ PO₄ and 2.5 mL ofNaOH.

c. PM: A buffer containing per 1 liter aqueous solution: 200 grams (g)sucrose, 0.25 g K₂ S)₄ in 890 mL H20, with the addition aftersterilization of 100 mL 0.25 M TES, pH7.2, 2 mL trace elements solution(Hopwood, et al, 1985, Genetic Manipulation of Streptomyces A LaboratoryManual, The John Innes Foundation), 0.08 mL 2.5 M CaCl2, 10 mL 0.5% KH₂PO₄, 2 ml 2.5M MgCl₂.

d. A4Bf: A growth medium containing per 1 liter aqueous medium: 15 g soyflour, 50 g glucose, 5 g NaCl, and 1 g CaCO₃.

e. SCM: A growth medium containing per 1 liter aqueous medium: 20 gsoytone, 15 g soluble starch, 10.5 g MOPS, 1.5 g yeast extract and 0.1 gCaCl₂.

The foregoing can be better understood by reference to the followingexamples, which are provided as non-limiting illustrations of thepractice of the instant invention. Both below and throughout thespecification, it is intended that citations to the literature beexpressly incorporated by reference.

EXAMPLE 1 Construction of plasmid pDPE4

DEP4 was constructed using standard methods of recombinant DNAtechnology according to the schematic outline shown in FIG. 2. A 2.55 kbEcoRI-PstI fragment containing eryK and flanking portions of ORF 19 andORF 21 was isolated from pEVEH8 and ligated to pUC18 cut with the sameenzymes to generate plasmid pDPE1. This plasmid was then cut withEco0109I and two of the three fragments generated (i.e. those havingsizes of 0.9 and 4.3 kb) were isolated. These two fragments were ligatedto generate pDPE2, which contains a small deletion within the eryK gene.A 2.1 kb EcoRI-PstI fragment from pDPE2 was then ligated to pCS5 cutwith the same enzymes to yield pDPE3. Additional contiguous DNA sequencewas added downstream of eryK by excising a 0.765 kb PstI fragmentcontaining ORF 19 from pEVEH8 and ligating this to PstI cut, CIAPtreated pDPE3, to generate pDPE4. Orientation was confirmed byrestriction analysis.

EXAMPLE 2 Construction of plasmid pGM504

pGM504 was constructed using standard methods of recombinant DNAtechnology according to the schematic outline shown in FIG. 3. pGM420, aStreptomyces-E. coli shuttle vector, was constructed by cutting pUC 18with SstI and ligating this plasmid into the SstI site of pIJ702. ThepUC 18 polylinker is oriented proximal to the BglII, SphI and Asp7l8sites of pIJ702. A 5.3kb PstI fragment of Sac. erythraea DNA containingeryF, flanking and nearby DNA including part of eryG was cloned into thePstI site of pGM420 to give pMW65. A 0.5 kb out of frame deletion ineryF was made by sequential partial digestions of pMW65 with Asp7l8 andSstI, and then filling in the sticky ends with pol1K and religating toyield pGM504.

EXAMPLE 3 Construction of plasmid pDEP35

pDPE35 was constructed using standard methods of recombinant DNAtechnology according to the schematic outline shown in FIG. 4. A 4.5 kbEcoRI-BamHI fragment from cosmid p7A2 (Paulus et al., J. Bacteriol.172:2541 (1990)) containing eryG was ligated to pBR322 cut with the sameenzymes, to give pGM403. The EcoRI-SphI fragment of pGM403 containingeryG was then ligated to pUC18 cut with the same enzymes to generatepDPE8. The ermE* promoter (carried on an EcoRI-BamHI fragment frompIJ4070) was inserted upstream of eryG into the EcoRI-BglII sites ofpDPE8 to create pKAS2. pKAS2 was digested to completion with EcoRI andthen partially with NaeI in order to isolate the 1.5 kb fragmentcontaining the ermE*-eryG fusion. This fragment was ligated to pUC18 cutwith EcoRI and HinclI to generate pKAS3. pKAS3 was digested with SspIand SphI to obtain a 3.4 kb fragment; this fragment was ligated to the0.6 kb fragment of pUC19 cut with the same enzymes in order to add anEcoRI site downstream of eryG to generate pKAS4.

The eryG gene was inserted into the Sac. erythraea DNA `second site`region in the following manner. An 11 kb HindIII fragment of Sacerythraea chromosomal DNA was ligated to a pBR322 derivative to generatepGM469. This HindIII fragment contains a unique StuI site into which wasinserted an EcoRI-StuI linker, to generate pGM473. This plasmid wasdigested with EcoRI and treated with CIAP. The 1.6 kb EcoRI fragmentfrom pKAS4 containing the ermE*-eryG fusion was isolated and ligated topGM473 to generate pKAS19. The 14 kb HindIII fragment of pKAS 19containing the `second site` region construct was then ligated to pCD1cut with HindIII and treated with CIAP to yield pKAS20.

The thiostrepton resistance gene was placed downstream of eryG in thefollowing manner. A 1.1 kb BclI fragment containing the tsr gene fromplasmid pCS5 was inserted into pUC19 (cut with BamHI and treated withCIAP) to generate pDPE23A. In order to insert a multiple cloning site(MCS) downstream of tsr, this plasmid was digested with EcoRI and ScaIand ligated to pUC18 cut with the same enzymes to give pDPE26. A 1.1 kbXbaI fragment containing tsr could then be isolated from pDPE26 andligated to XbaI cut and CIAP treated pKAS20 to generate pDPE34.

Removal of the second copy of tsr from pDPE34 was accomplished in thefollowing manner. The 3 kb NdeI-EcoRI fragment from pCD1 containing aSac. erythraea origin of replication was ligated to pUC19 digested withthe same enzymes to give plasmid pDPE21. The 15 kb HindIII fragment ofSac. erythraea DNA containing the ermE* promoter, eryG and tsr frompDPE34 was then ligated into the HindIII site of pDPE21 to give plasmidpDPE35.

EXAMPLE 4 Construction of Sac. erythraea eryK, eryF strain (ER720-KF)

An example of a 6,12-dideoxyerythromycin A producing microorganism wasprepared by replacing the wild type eryK and eryF of Sac. erythraeaER720 cells with deletions in these genes carried on the recombinantplasmids of Examples 1 and 2. Transformation and resolution of theintegration event was carried out according to the following method.Sac. erythraea ER720 cells were grown in 50 mL of SGGP medium for 3days, at 32° C. and then washed in 10 mL of 10.3% sucrose. The cellswere resuspended in 10 mL of P_(M) buffer containing 1 mg/mL lysozymeand incubated at 30° C. for 15-30 minutes until most of the mycelialfragments were converted into spherical protoplasts. The protoplastswere washed once with P_(M) and then resuspended in 3 ml of the samebuffer containing 10% DMSO for storage in 200 mL aliquots at -80° C.

Transformation was carried out by quickly thawing an aliquot ofprotoplasts, centrifuging for 15 seconds in a microfuge, decanting thesupernatant, and resuspending the protoplasts in the PM remaining in thetube. Ten μL of DNA solution was added (3 μL of pDPE4 DNA from Example Iat about 1 μg/μL in 7 μL of P_(M) buffer) and mixed with the protoplastsby gently tapping the tube. Two tenths of a mL of 25% PEG 8000 in Tbuffer (Hopwood, et al, 1985, Genetic Manipulation of Streptomyces ALaboratory Manual, The John Innes Institute) was then added, mixed bypipetting the solution three times and the suspension immediately spreadon a dried R3M plate. The plate was incubated at 30° C. for 20 hours andoverlayed with 2 mL of water containing 100 μg/mL thiostrepton, driedbriefly and incubated 4 more days at 30° C.

To select integrants, transformants were replica plated ontonon-selective R3M medium (i.e. without thiostrepton), allowed tosporulate and then replica plated onto R3M medium containing 10 μg/mLthiostrepton. 10 colonies were inoculated into SGGP containingthiostrepton. Of these, 8 grew and were selected as integrants.Integration of the plasmid DNA was confirmed by Southern hybridization,and all 8 strains were found by TLC analysis to make erythromycin A.

The 8 integrants were then grown non-selectively on R3M and allowed tosporulate. Spores were plated to obtain individual colonies on R3Mplates, which were then screened for sensitivity to thiostrepton,indicating loss of the plasmid sequence from the chromosome. Eightthiostrepton sensitive colonies were selected and two of these wereconfirmed by Southern hybridization and by the production ofeiythromycins D and B to contain the deleted copy of eryK in thechromosome.

Replacement of eryF with a deleted copy was performed as described abovefor the eryK deletion, except that the eryK deleted strain was used asthe recipient of pGM504 (described in Example 2). Integration andexcision of the plasmid from the Sac. erythraea chromosome was monitoredby Southern analysis, and the resulting strain, named ER720-KF, wasfound to produce a mixture of 6,12-dideoxyerythromycin A and6-deoxyerythromycin D.

EXAMPLE 5 Construction of Sac. erythraea eryK, eryF, `second site`::eryG+(ER720-KFG+)

A preferred example of the 6,12-dideoxyerythromycin A producingmicroorganism of the present invention was prepared by transformingER720-KF cells with the recombinant plasmid of Example 3 (i.e. pDPE35)to construct a strain which produces highly pure 6,12-dideoxyerythromycin A rather than a mixture of 6,12-dideoxyerythromycinA and 6-deoxyerythromycin D. Integration and excision of pDPE35 from theSac. erythraea chromosome to leave behind a second copy of eryG drivenby the ermE* promoter was performed as follows. Protoplasts of ER720-KFcells were transformed with pDPE35 as described in Example 4. In orderto resolve the duplication created by the integration of the plasmid ata region of homology of unknown but non-essential function in the Sac.erythraea chromosome, and as a result leave behind the eryG and thethiostrepton resistance marker carried by that plasmid, transformantswere streaked two consecutive times on R3M plates containingthiostrepton. Those colonies which were able to grow after two passageson thiostrepton were found by Southern analysis to contain a second copyof eryG integrated into the `second site` region of the chromosome. Thestrain was designated ER720-KFG+.

EXAMPLE 6 Fermentation of ER720-KF and ER72OKFG+, and Identification ofCompounds Produced by the 2 Strains

The recombinant Sac. erythraea strains produced in Examples 4 and 5 werecultivated using the following fermentation procedure. Six hundred mLseed cultures of ER720-KF and ER72OKFG+ were grown in A4Bf medium incotton plugged 2-liter flasks at 32° C., at 225 rpm for 48 and 72 hours,respectively. Forty-five liter LH fermenters (Incel Tech, Hayward,Calif.) containing 30 liters of SCM medium (with thiostrepton added to10 μg/mL for ER720-KFG+) were inoculated with 1.5 liters of seedculture. Cells were grown at 32° C., at 250 rpm with a head pressure at5 psi, and an aeration rate of 0.7-1 volumes of O_(2/) volume ofculture/minute. Antifoam was added to 0.01% initially and pH wascontrolled at 7.0 with propionic acid and KOH. Culture samples weretaken at 0, 24, 40, 48, 66, 72 and 144 hours for ER720-KF and 24, 40,48, 66, 72, 88 and 144 hours for ER720-KFG+.

Erythromycin derivatives were isolated from the culture broth of theproducing strains by the following procedure. Cells were removed from1.5 mL of culture by centrifugation for one minute in a microfuge. OnemL of the supernatant was removed to another tube and the pH adjusted to9.0 by the addition of 6 μL NH₄ OH. One half mL of ethyl acetate wasadded, the tube was vortexed for 10 sec and then centrifuged forapproximately 5 minutes to separate the phases. The organic phase wasremoved to another tube, and the aqueous phase was re-extracted with 0.5mL of ethyl acetate. The second organic phase was pooled with the firstand dried in a Speed Vac. The residue was taken up in 11 μL of ethylacetate and 1 μL was spotted onto TLC plates. A standard curve of6,12-dideoxyerythromycin A was also included to insure that the amountsof compound applied to the plate were in the linear range of thedetection method.

Silica gel thin-layer chromatography plates (Merck 60F-254) weredeveloped using isopropyl ether-methanol-NH₄ OH (75:35:2). Compoundswere visualized by spraying the plates with anisaldehyde-sulfuricacid-ethanol (1:1:9). With this reagent, 6,12-dideoxyerythromycin A and6-deoxyeiythromycin D appear as blue spots and are additionallyidentified by comparing their R_(f) values (ratio of movement of thespot to the movement of the solvent front) with that of standards (seeFIGS. 6 and 7).

The ratio of 6,12-dideoxyerythromycin A to 6-deoxyerythromycin Dproduced by the genetically engineered strains was analyzed by measuringTLC spots with a Molecular Dynamics Personal Densitometer (PD-120 laserbased transmission scanner) at 100 μm resolution. FIG. 8a demonstratesthat over a 6 day fermentation, while the strain lacking the C-6 andC-12 hydroxylases produced 6,12-dideoxyerythromycin A, it alsoaccumulated a large amount of the non-methylated precursor,6-deoxyeiythromycin D. However, as shown in FIG. 8b, when an extra copyof the 3"-O-methyltransferase gene was added to a non-essential regionof this strain, it was able to overcome the accumulation of6-deoxyerythromycin D, and convert this precursor to highly pure6,12-dideoxyerythromycin A.

EXAMPLE 7 Construction of plasmid pKAS37

pKAS37 was constructed using standard methods of recombinant DNAtechnology according to the schematic outline in FIG. 9. The ermE*promoter from pIJ4070 was inserted into the BamHI/EcoRI sites of pUC19,to give pKAS7. A region of the polylinker including the KpnI to BglIIsites was moved from pIJ4070 to pUC19 to give pKAS8. pKAS7 was digestedwith SspI/BamHI and the ermE* promoter fragment inserted into SspI/BglIIdigested pKAS8 to give pKAS 16. To eliminate a PvuII site, plasmid pKAS16 was digested with NaeI/EcoO109, filled-in with Klenow and ligated togenerate pKAS17. pKAS17 was then digested with SspI/HindIII and theermE* fragment ligated into a similarly digested pIJ4070 to give pKAS18.

The tsr gene was excised from pCS5 by BclI digestion and ligated intoBamHI digested pUC19 dephosphorylated with CIAP to give pDPE23B. pDPE23Bwas then digested with SspI/NdeI and the ermE* fragment was isolatedfrom pKAS18 digested with SspI/AseI and ligated to generate pKAS23.pKAS23 was digested with SstII/PvuII and the 1.1 kb SstII/SmaI fragmentfrom pCS5 were ligated to give pKAS33.

pDPE35 was digested with HindIII/KpnI and the pCD1 replicon fragmentligated to a similarly digested pGM469 to give pKAS30. pKAS30 wasdigested separately with HindIII and KpnI with concurrent fill-in withKlenow to give pKAS34. pKAS34 was partially digested with StuI,dephosphorylated with CIAP and the fragment containing ermE* and tsrgene from pKAS33 digested with SspI/FspI were ligated to generatepKAS35(-). pDPE36 was generated by digesting pDPE21 with MluI/Ndel,filling-in with Klenow and ligating. pKAS35(-) was digested withEcoRI/StuI and the pCD1 replicon from pDPE36 similarly digested wereligated to generate pKAS36. pKAS36 and pkAS35(-) were digested withSspI/EcoRI to generate pKAS37. A detailed restriction map of thisplasmid is shown in FIG. 10. A culture of E. coli DH5α which containsplasmid pKAS37 has been deposited as above with the AgriculturalResearch Culture Collection Peoria Ill. under the tern of the BudapestTreaty and has been accorded the accession number NRRL B-21485.

EXAMPLE 8 Construction of plasmid pKASI37

pKASI37, an alternative embodiment of pKAS37, is constructed usingstandard methods of recombinant DNA technology according to theschematic outline in FIG. 11. To obtain the `second site` region ofpKASI37, Sac. erythraea ER720 or NRRL2338 chromosomal DNA is digestedwith HindIII and fragments of approximately 12 kb are isolated from a0.7% agarose gel. This pool of fragments is ligated to pUC19 (alsodigested with HindIII). Transformants are screened for plasmids carryingthe second site by digestion of miniprep DNA with BamHI, MluI and StuIto generate expected fragments of 6.8, 5.6 and 2.1 kb (for BamHI), 11.3,3.3 and 0.3 kb (for MluI) and 14.9 kb (for StuI). Proper orientation ofthe HindIII fragment is determined by KpnI digestion as the KpnI site atone end of the `second site` region should be adjacent to the KpnI sitein the pUC19 polylinker. The resulting plasmid is pX1.

Plasmid pCD1 is then digested with MluI, treated with Klenow anddigested with KpnI. The resulting fragment of about 3 kb containing theSac. erythraea replicon is ligated to pX1 digested with NdeI (filled inwith Klenow) and KpnI to form plasmid pX2. Plasmid pX2 is then digestedwith KpnI, treated with Klenow and religated to give plasmid pX3. pX3 isdigested with HindIII, treated with Klenow and religated to give plasmidpX4.

The final construction steps of the plasmid involve insertion of theermE* promoter, polylinker and tsr gene. Plasmid pIJ4070 is digestedwith KpnI, treated with Klenow and religated to form plasmid pX5. Twooligonucleotides are then synthesized which when annealed will containthe following restriction sites:BgllI-EcoRI-KpnI-XbaI-HindIII-BglII-BamHI-EcoRI-PstI (synthesizedpolylinker). This double stranded fragment is ligated into pX5 digestedwith BamHI and PstI to give plasmid pX6. Plasmid pCS5 is then digestedwith BclI and the resulting 1 kb tsr containing fragment is ligated intothe BamHI site of pX6 to give plasmid pX7 pX7 is then partially digestedwith EcoRI, treated with Klenow and the 1.4 kb DNA fragment containingthe ermE* promoter-synthesized polylinker-tsr gene is inserted into theunique StuI site of the Sac. erythraea `second site` region in pX4 toform plasmid pKASI37. A detailed restriction map of plasmid pKASI37 isshown in FIG. 12.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES:1                                                   (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 917 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: genomic DNA                                               (iii) HYPOTHETICAL: No                                                        (v) FRAGMENT TYPE: internal                                                   (vi) ORIGINAL SOURCE: Saccharopolyspora erythraea                             (ix) FEATURE:                                                                 (A) NAME/KEY: 1kb portion ofsecond site region                                (B) LOCATION:                                                                 (C) OTHER INFORMATION: Non-essential function                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GAGCGACCACAGGTGGGCCCGGATGTTGCAGCCTTGGTCGGGGTAGTCGATGCGGATTCG60                GAACAGTGCCACGGCTGTGGTGTTCGAAGGTGGAAGTCTTGAGCTGCTGGTGCCACCGGA120               TTGCTTGCTCCAGCGAGACCGCGTTGCCGTTGACGAAGGCCAACGCGTCAAACACCGCCT180               GGGAGTGCTCGGGTCGCAGTTTCTTCAAGTCATCGCTGAGAATCCCGGCACCGAGCGTGA240               TAGGCATCCTGCACCGCCCCACACGGCGCGGAGATTGCGGTCCAGGCCCGGCAACATACC300               AGCGCTTCGTCGAACTCGTCCGCCTCGACGTGGGCCCGCAGTTGTTCCGCGAACACTGCG360               CAGTTCGGAGCAGCTTCTGGCCCAGGGCTTGCGACAACCTTGGGTGGGGTGTGCGCGGGG420               TTGGTGCTGAAGTCGTTGCGGAAACCCAGCATCGTCAGAGCGTGGTCGAACTGTGCTGGA480               CTGAGGTGCTCAGACAGCACACGAATCCAGCTCCCTGCCGGTGTGCTGCCAGAAGGGGAC540               CGCGAGGCCCGCGGAATCTCCGCCGGATCGCCCCGAAGCCGACCCAGCTCACGCAACACC600               GAATCGGTGTCCGGCCGAGGTGACCGTGTGCCCGACCCGGAGCCGGGAGCACGCCGCGCA660               CTGGGCCTCCTCGGTTGTGTGTGTGAGATCGTCGTTCCTCGAATTTAAGCAAGCCGGCGA720               TGAACTTCGCCCGGCGCGCGGACAACGTCGTCACATCACCGTCCGCCCCGACGCCAGAAG780               CCGAGCCAGCCCCCGCACTGCGGCCCGAACGGAACCTCCTCGGAAGTTACGCCGGAGCTG840               CCCGGTGCCGCCGTGGTCAGGAAAGCCTGCGCGTGCTGAGGGAGCCGTCCATGTTGATAA900               TTATTATCTCAGATGAC917                                                          __________________________________________________________________________

We claim:
 1. A recombinant DNA vector for integrating a gene of interestinto the chromosome of an erythromycin producing host cell, said vectorcomprising a first DNA sequence having an 11 kb HindIII fragment of theSaccharopolyspora erythraea chromosome wherein said DNA sequencecontains SEQ ID NO:1, a second DNA sequence which contains the origin ofreplication from plasmid pCD1, and a third DNA sequence encoding aselectable maker gene.
 2. The DNA vector of claim 1 further comprising aDNA sequence which encodes the ermE* promoter wherein said ermE*promoter is operably linked to said gene of interest.
 3. The DNA vectorof claim 2 wherein said gene of interest is the eryG gene.
 4. The DNAvector of claim 1 further comprising a multiple cloning site.
 5. The DNAvector of claim 1 wherein said vector is plasmid pKAS37.
 6. The DNAvector of claim 5 further comprising the eryG gene.
 7. The DNA vector ofclaim 1 wherein said vector is plasmid pDPE35.
 8. A method forincreasing 3"-O-methyltransferase activity in host cells which produceat least one substrate for said 3"-methyltransferase activity, saidmethod comprising:a. introducing an integrative recombinant vectorcomprising a first DNA sequence having an 11 kb HindIII fragment of theSaccharopolyspora erythraea chromosome for integrating said vector intothe chromosome of said host cells wherein said DNA sequence contains SEQID NO: 1, a second DNA sequence which contains the origin of replicationfrom plasmid pCD1, a third DNA sequence encoding a selectable makergene, and a fourth DNA sequence which is the eryG gene, said eryG geneencoding said 3"-O-methyltransferase activity; and b. selecting forstable integrants of said host cells, said integrants having said eryGgene stable integrated into said chromosome of said integrants.
 9. Themethod of claim 8 wherein said integrative recombinant vector is theplasmid of claim
 7. 10. The method of claim 8 wherein said integrativerecombinant DNA vector is the plasmid of claim
 8. 11. A method formaking a modified deoxyerythromycin producing host strain, wherein saidmodification is an increase in 3"-O-methyltransferase activity, saidmethod comprising:a. introducing an integrative recombinant vectorcomprising a first DNA sequence having an 11 kb HindIII fragment of theSaccharopolyspora erythraea chromosome for integrating said vector intothe chromosome of said host cells, wherein said DNA sequence containsSEQ ID NO:1, a second DNA sequence which contains the origin ofreplication from plasmid pCD1, a third DNA sequence encoding aselectable maker gene, and a fourth DNA sequence which is the eryG gene,said eryG gene encoding said 3"-O-methyltransferase activity; and b.selecting for stable integrants of said host cells, said integrantshaving said eryG gene stable integrated into said chromosome of saidintegrants.
 12. The method of claim 8 wherein said integrativerecombinant vector is the plasmid of claim
 7. 13. The method of claim 11wherein said integrative recombinant DNA vector is the plasmid of claim8.
 14. A host strain which produces an erythromycin derivative, saidhost strain being modified to overproduce 3"-O -methyltransferaseactivity wherein said modification is achieved by the method of claim 8.